Soil bacteria of the genus Rhizobium, a member of the family Rhizobiaceae, are capable of infecting plants and inducing highly differentiated structures, root nodules, within which atmospheric nitrogen is reduced to ammonia by the bacteria. The host plant, most often of the family Leguminosa, utilizes the ammonia as a source of nitrogen. Previously, Rhizobium species were classified in two groups, either as fast-growing or slow-growing. The group of slow-growing rhizobia has recently been reclassified as a new genus designated Bradyrhizobium (Jordan, D.C. (1982) Int. J. Syst. Bacteriol. 32:136). Included in the classification of fast-growing rhizobia are R. trifolii, which nodulate clover, R. melitoti, which nodulate alfalfa, R. leguminosarum, which nodulate pea, and R. phaseolus, which nodulate bean. These fast-growing strains of rhizobia generally display narrow host range. The fast-growing R. japonicum strains which nodulate Glycine max cv. Peking and fast-growing members of the cowpea Rhizobium display broader host range. The Bradyrhizobium, slow-growing rhizobia, include the commercially important soybean nodulating strains B. japonicum (i.e., strains USDA 110 and 123), the slow-growing promiscuous rhizobia of the cowpea group, and B. parasponia (formerly Parasponia Rhizobium) which nodulates the non-legume Parasponia, as well as a number of tropical legumes including cowpea and siratro.
Nodulation and the development of effective symbiosis is a complex process requiring both bacterial and plant genes. Several recent reviews of the genetics of the Rhizobium-legume interaction are found in Broughton, W. J., ed. (1982) Nitrogen Fixation, Volumes 2 and 3 (Clarendon Press, Oxford); Puhler, A. ed. (1983) Molecular Genetics of the Bacteria-Plant Interaction (Springer-Verlag, Berlin); Szalay, A. A. and Leglocki, R. P., eds. (1985) Advances in Molecular Genetics of the Bacteria-Plant Interaction Cornell University Publishers, Ithaca, N.Y.; Long, S. R. (1984) in Plant Microbe Interactions Volume 1, Kosuge, T. and Nester, E. W., eds. (MacMillan, N.Y.) pp. 265-306; and Verma, D. P. S. and Long, S. L. (1983) International Review of Cytology (Suppl. 14), Jeon, K. W. (ed.), Academic Press, p. 211-245.
In fast-growing species genes required for nodulation and nitrogen fixation are located on large Sym plasmids. Although the process of recognition, infection and nodule development is complex, it appears that at least for the fast-growing rhizobia relatively few bacterial genes are directly involved, and these are closely linked on the Sym plasmid. For example, a 14 kb fragment of the R. trifolii Sym plasmid is sufficient to confer clover-specific nodulation upon a Rhizobium strain cured of its Sym plasmid (Schofield et al. (1984) Plant Mol. Biol. 3:3-11). Nodulation and nitrogenase genes are localized on symbiotic plasmids in R. leguminosarum (Downie et al (1983) Mol. Gen. Genet. 190:359-365) and in R. meliloti (Kondorosi et al. (1984) Mol. Gen. Genet. 193:445-452). In contrast, no Sym plasmids have been associated with the slow-growing rhizobia, B. japonicum or B. parasponia. The nitrogenase and nodulation genes of these organisms are believed to be encoded on the chromosome.
Fine structure genetic mapping has been used to locate individual nodulation genes in both fast- and slow-growing rhizobia. Transposon mutagenesis, most often using the transposon Tn5, has identified about 10 nodulation genes associated with non-nodulation and delayed nodulation phenotypes (Djordjevic et al. (1985) Mol. Gen. Genet. 200:263-271; Downie et al. (1985) Mol. Gen. Genet. 198:255-262; Kondorosi et al., 1984 and Innes et al. (1985) Mol. Gen. Genet. 201:426-432). Common nodulation genes designated nodABC and D, which are functionally and structurally conserved among the fast-growing rhizobia, have been identified by hybridization studies and cross-species complementation experiments. Both B. parasponia (Marvel et al. (1985) Proc. Natl. Acad. Sci. USA 82:5841-5845) and B. japonicum (Russel et al. (1985) J. Bacteriol. 164:1301-1308) contain nodulation genes which can functionally complement mutations in fast-growing rhizobia and which show strong structural homology to nodulation gene regions of R. meliloti and R. leguminosarum. Recent work (Rolfe et al. (1985) Nitrogen Fixation Research Progress, Evans et al. (eds.) Martinus Nijhoff, Dordrecht, The Netherlands, p. 79-85; Scott et al. (1985) ibid., p. 130; and Schofield et al. (1985) ibid., p. 125) reports that the structure, organization and regulation of the common nod genes is conserved in both fast- and slow-growing rhizobia. Structural and functional conservation extends to other nodulation genes (nodE, F, I and J) (Djordjevic et al., (1985) Plant Mol. Biol. 4:147-160).
In the common nod region, nodA, B and C genes are grouped sequentially and are likely to be coordinately transcribed as a single transcriptional unit. The nodD gene is transcribed divergently from the nodABC operon (Egelhoff et al. (1985) DNA 4:241-248; Jacobs et al. (1985) J. Bacteriol. 162:469-476; Rossen et al. (1984) Nucl. Acids Res. 12:9497-9508; and Torok et al. (1984) Nucl. Acids Res. 12:9509-9524). Divergent promoters for the nodABC operon and nodD are presumably located in the region between nodD and nodA. In R. trifolii, at least (see FIG. 1), nodI and nodJ are believed to be cotranscribed with nodABC, as a nodABC(IJ) operon while nodE and F form a nodEF operon. Both nodG and nodH are believed to be transcribed independently.
The manner in which the nodulation genes are regulated is also conserved among Rhizobium and Bradyrhizobium strains. Only one of the nodulation genes, nodD, is constitutively expressed in free-living rhizobia. Expression of the other identified nodulation genes including nodABCEFGHI and J requires the presence of legume exudate and the gene product of nodD (Mulligan and Long (1985) Proc. Natl. Acad. Sci. USA 82:6609-6613; Rossen et al. (1985) EMBO J. 4:3369-3373; Innes et al., 1985). Conserved DNA sequence elements are found within the promoter regions of the nodABC(IJ) operon in R. trifolii, R. meliloti, R. leguminosarum and B. parasponia, and in the nodEF operon and nodH in R. trifolii. (Scott et al. (1985); Rolfe et al. (1985); Kondorosi et al. (1985) Nitrogen Fixation Research Progress, Evans et al. (eds.) Martinus Nijhoff, Dordrecht, The Netherlands, p. 73-75). These consensus sequences are associated with exudate-inducible expression of the nod genes and their presence in various rhizobia suggest a conserved regulatory mechanism.
Sym plasmid encoded nodulation genes of Rhizobium strains and their analogues in Bradyrhizobium strains affect the early stages of nodule formation including host-bacterium recognition, infection and nodule development. Individual wild type strains of Rhizobium and Bradyrhizobium species display some variation in these early nodulation steps. This variation is reflected in differences in relative rates of initiation of nodulation and ultimately in differences in competitiveness between strains for nodule occupancy. Strains which initiate infection and nodules earlier will occupy a greater portion of the nodules on a given plant. Improving the competitiveness of a specific Rhizobium is an important part of the development of improved inocula for legumes. A more effective Rhizobium strain, which would likely constitute an improved inoculum, must be able to out-compete the indigenous rhizobia population for nodule occupancy in order for their improved qualities to impact on the inoculated legume. An inoculating composition or an inoculating method which would improve competitiveness of a selected inoculant strain is therefore of significant commercial importance.
The establishment of nitrogen-fixing nodules is a multistage process involving coordinated morphological changes in both bacterium and plant requiring precise regulation of plant and Rhizobium genes. It has been suggested that an exchange of signals between plant and bacterium is requisite for mutual recognition and coordination of the steps of infection and nodulation development (Nutman, P. S. (1965) in Ecology of Soil Borne Pathogens, eds. F. K. Baker and W. C. Snyder, University of California Press, Berkeley, pp. 231-247; Bauer, W. D. (1981) Ann. Rev. Plant Phys. 32:407-449; and Schmidt, E. E. (1979) Ann. Rev. Microbiol. 33:355-376). The regulation of nodulation genes of rhizobia by chemical factors excreted by legumes in exudates is a specific demonstration of communication between host and symbiont.
Legume exudates have been previously linked to both stimulation (Thornton (1929) Proc. Royal Soc. B 164:481; Valera and Alexander (1965) J. Bacteriol. 89:1134-1139; Peters and Alexander (1966) Soil Science 102:380-387) and inhibition (Turner (1955) Annals Botany 19:149-160; and Nutman (1953) Annals Botany 17:95-126) of nodulation by rhizobia.
Turner (1955) reported that addition of activated charcoal to rooting medium of clover plants led to an increased rate of nodule initiation. Activated charcoal was demonstrated to remove by adsorption an unidentified inhibitory substance secreted by clover roots. Nutman (1953) reported that clover roots excreted a substance inhibitory to nodulation. The substance was not identified but was found to be associated with the stage of nodulation on the plant. In both cases, it was suggested that both stimulatory and inhibitory factors were present in the root exudate.
Valera and Alexander (1965) and Peters and Alexander (1966) reported a nodulation enhancing factor in legume exudates that was dialyzable, water soluble and thermostable. This factor was replaceable by coconut water. More recently, Baghwat and Thomas (1982) Applied Environ. Microbiol. 43:800-805 described a stimulatory factor from legume exudates that was thermostable, was high molecular weight (about 2.times.10.sup.5) and was composed of protein and neutral hexoses. This factor was associated with elimination of nodulation delay in a certain cowpea Rhizobium strain. Halverson and Stacey (1984) Plant Physiol. 74:84-89; and (1985) Plant Physiol. 77:621-624 reported an exudate factor having a similar effect on nodulation initiation in B. japonicum USDA 110 mutants. In contrast to Baghwat and Thomas (1982), this stimulator of nodulation is described as a heat and trypsin sensitive protein, a galactose-specific lectin.
In addition to factors present in legume exudate, diverse chemicals have been identified as stimulators or inhibitors of nodulation. Reported stimulators of nodulation include inositol, indole, 2-phenol-n-butyric acid, D-leucine, barbituric acid, pyridine-3-sulfonate and quercetin (Molina and Alexander (1967) Can. J. Microbiol. 13:819-827; and Weir (1960) Phyton 15:109-118).